Phospholipase D gene originated from plant

ABSTRACT

PCT No. PCT/JP94/01627 Sec. 371 Date Jul. 26, 1995 Sec. 102(e) Date Jul. 26, 1995 PCT Filed Sep. 30, 1994 PCT Pub. No. WO95/09234 PCT Pub. Date Apr. 6, 1995A cloned DNA encoding phospholipase D originated from a plant and a cloned DNA which regulates expression of phospholipase D gene originated from a plant are disclosed.

TECHNICAL FIELD

The present invention relates to a phospholipase D gene originated from a plant.

BACKGROUND ART

Phospholipase D (hereinafter also referred to as "PLD") is one of phospholipid-decomposing enzymes, which catalyzes, for example, the reaction of decomposing lecithin to liberate phosphatidic acid and choline. This enzyme is known to occur in plants, animals and microorganisms. PLD is used as a reagent for measuring phospholipids in blood, as well as for hydrolysis of phospholipids and for production of derivatives by a base-exchange reaction utilizing the reversible reaction by PLD.

Purification and partial purification of PLDs originated from plants have been reported. That is, purification of rice PLD (Men Hui Lee, 1989, Phospholipase D of rice bran, I, purification and characterization, Plant Science 59, 25-33); partial purification of rice PLD (Katsumi TAKANO, Ikuzo KAMOI and Tetsujiro OBARA, 1987, About separation and purification, and properties of rice bran phospholipase D, J. Jpn. Soc. Food Sci. Technol., 34, 8-13 (1987)); purification of peanuts PLD (Michael Heller, Nava Mozes, Irena Peri and Eddie Maes, 1974, Phospholipase D from peanut seeds, IV, Final purification and some properties of the enzyme, Biochem. Biophys, Acta 369, 397-410); and purification of cabbage PLD (Romy Lambrecht and Renate Ulbrich-Hofmann, 1992, A facile purification procedure of phospholipase D from cabbage and its characterization, Biol. Chem. Hoppeseyler 373(2), 81-88) have been reported. However, amino acid sequences of plant PLDs have not been reported at all and genetical analyses thereof have also not been reported.

On the other hand, analyses of PLD genes of microorganisms and animals have been reported (Japanese Laid-open Patent Application (Kokai) No. 3-187382; Adrian L. M. Hodgson, Phillip Bird, and Ian T. Nisbet 1990, Cloning, nucleotide sequence, and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis, Journal of Bacteriology 172, 1256-1261; and Japanese Laid-open Patent Application (Kokai) No. 5-76357).

If a PLD gene originated from a plant were available, the PLD originated from the plant may be produced in a large scale by a genetic engineering process, which is industrially advantageous. However, as mentioned above, since the amino acid sequence and DNA sequence of plant PLD have not been reported, it has hitherto been impossible to genetically manipulate the PLD gene. Further, as mentioned above, although the amino acid sequences and DNA sequences of PLDs of microorganisms and animals have been reported, since homologies of sequences are not observed among the PLD genes of microorganisms or among the PLD genes of microorganisms and animals, it is difficult to isolate plant PLD gene based on the reported information. Further, since there is no information about plant PLD gene, an antisense DNA which suppresses expression of the plant PLD gene has not been obtained.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a PLD gene originated from a plant. Another object of the present invention is to provide an antisense DNA which can suppress the expression of the above-mentioned PLD gene according to the present invention. Still another object of the present invention is to provide a DNA which regulates expression of the PLD gene originated from a plant.

After intensive study, the present inventors succeeded in isolation of rice PLD gene and in sequencing the PLD gene by purifying PLD from rice, determining the partial amino acid sequence thereof, screening a rice cDNA library using as a probe the PCR product obtained by using the oligonucleotides encoding the above-mentioned partial amino acid sequence, cloning the inserted gene of positive clones and sequencing the inserted gene. Further, the present inventors succeeded in obtaining a cDNA clone of maize PLD using as a probe the DNA encoding rice PLD, and in sequencing the maize PLD gene. The present inventors still further succeeded in isolating a genome DNA clone carrying the regulatory sequence of rice PLD gene and in sequencing it.

That is, the present invention provides a DNA encoding phospholipase D originated from a plant. The present invention also provides a DNA which regulates the expression of phospholipase D gene originated from a plant.

By the present invention, a PLD gene originated from a plant was cloned for the first time. By using the DNA according to the present invention, the PLD originated from a plant, which is industrially useful, can be produced in a large scale by a genetic engineering process. Further, by the present invention, a DNA which regulates expression of the PLD gene originated from a plant was cloned for the first time. By virtue of this, expression of lipid-related genes may be suppressed, thereby modifying plant lipids.

BEST MODE FOR CARRYING OUT THE INVENTION

As mentioned above, the DNA according to the present invention encodes PLD originated from a plant. In the examples hereinbelow described, the DNA encoding rice PLD was isolated and sequenced. The deduced amino acid sequence encoded by the thus sequenced DNA is shown in SEQ ID NO. 2 in the Sequence Listing. The experimentally determined nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 1. Therefore, the amino acid sequence shown in SEQ ID NO. 1 is identical to the amino acid sequence shown in SEQ ID NO. 2. Further, in the examples hereinbelow described, a cDNA clone of maize PLD gene was isolated using as a probe the thus sequenced DNA encoding the rice PLD and the maize PLD gene was sequenced. The deduced amino acid sequence encoded by the thus sequenced DNA is shown in SEQ ID NO. 4 in the Sequence Listing. The experimentally determined nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 4 is shown in SEQ ID NO. 3. Therefore, the amino acid sequence shown in SEQ ID NO. 3 is identical to the amino acid sequence shown in SEQ ID NO. 4.

Further, in the examples described below, rice cDNA library was screened using as a probe the thus obtained DNA encoding rice PLD and positive clones were isolated, followed by sequencing the inserted DNA to obtain a clone carrying an inserted DNA having the nucleotide sequence shown in SEQ ID NO. 5. In SEQ ID NO. 5, the 1876th base is "A" which is the first "A" in the translation initiation codon ATG of the PLD structural gene, and the region upstream of the "A" (i.e., 1-1875nt) is thought to be a regulatory region of the PLD gene. The amino acid sequence encoded by the region downstream of 1876nt is interrupted by introns.

The DNA according to the present invention may be obtained by, for example, the method described in the examples below in which rice or maize is used as the starting material. Alternatively, since the nucleotide sequence of the gene was disclosed by the present invention, the PLD gene may easily be prepared by PCR method using as primers a pair of oligonucleotides each of which hybridizes with the respective end of the DNA according to the present invention and using the genome DNA of rice or maize as a template.

A plant producing PLD may be obtained by inserting the DNA encoding PLD originated from a plant according to the present invention into a vector for plants by a conventional method and transforming a plant with the obtained recombinant vector by a conventional method.

By inserting the DNA according to the present invention into a vector for plants in the reverse direction, an antisense DNA of PLD gene may be obtained. By transforming a plant with such a recombinant vector, the mRNA which hybridizes with the mRNA formed by transcription of the PLD gene intrinsic to the plant is produced, so that expression of the PLD gene intrinsic to the plant may be suppressed. If this technique is applied to a rice plant, a rice plant in which expression of PLD gene is suppressed is obtained, so that the taste of the rice is improved.

The DNA regulating the PLD gene originated from a plant according to the present invention may be used in combination with the antisense DNA of the PLD gene. By this, the antisense DNA can be expressed at the same place as the place in which the PLD gene intrinsic to the plant is expressed.

It is well-known in the art that there are cases wherein the activity of an enzyme is retained even if the amino acid sequence of an enzyme is modified to a small extent, that is, even if one or more amino acids in the amino acid sequence are substituted or deleted, or even if one or more amino acids are added to the amino acid sequence. DNAs encoding the proteins having such modifications and having PLD activity are included within the scope of the present invention. That is, cloned DNAs encoding amino acid sequences having the same amino acid sequence as SEQ ID NO. 2 or SEQ ID NO. 4 except that one or more amino acids are substituted, deleted or added, which give the enzyme activity of PLD, are also included in the scope of the present invention. Similarly, DNAs having the same nucleotide sequence as SEQ ID NO. 5 except that one or more nucleotides are substituted, deleted or added, which regulate expression of the DNA encoding the amino acid sequence giving the enzyme activity of PLD are also included within the scope of the present invention.

Modification of DNA which brings about addition, deletion or substitution of the amino acid sequence encoded thereby can be attained by the site-specific mutagenesis which is well-known in the art (e.g., Nucleic Acid Research, Vol. 10, No. 20, p6487-6500, 1982). In the present specification, "one or more amino acids" means the number of amino acids which can be added, deleted or substituted by the site-specific mutagenesis.

Site-specific mutagenesis may be carried out by, for example, using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA except that the desired mutation as follows. That is, using the above-mentioned synthetic oligonucleotide as a primer, a complementary chain is produced by a phage, and host bacterial cells are transformed with the obtained double-stranded DNA. The culture of the transformed bacterial cells is plated on agar and plaques are formed from a single cell containing the phage. Theoretically, 50% of the new colonies contain the phage having a single-stranded chain carrying the mutation and remaining 50% of the colonies contain the phage having the original sequence. The obtained plaques are then subjected to hybridization with a kinase-treated synthetic probe at a temperature at which the probe is hybridized with the DNA having exactly the same sequence as the DNA having the desired mutation but not with the original DNA sequence that is not completely complementary with the probe. Then the plaques in which the hybridization was observed are picked up, cultured and the DNA is collected.

In addition to the above-mentioned site-specific mutagenesis, the methods for substituting, deleting or adding one or more amino acids without losing the enzyme activity include a method in which the gene is treated with a mutagen and a method in which the gene is selectively cleaved, a selected nucleotide is removed, added or substituted and then the gene is ligated.

EXAMPLES

The present invention will now be described more concretely by way of examples. However, the present invention is not restricted to the examples described below.

1. Purification of rice bran PLD

For the purification, a reference relating to the purification of rice bran PLD (Takano et al., J. Jpn. Soc. Food Sci. Technol., 34, 8-13 (1987)) was referred. The enzyme activity was measured by using phosphatidylcholine as the substrate and by quantifying the choline generated by the enzyme reaction by the choline oxidase method (Imamura et al., J. Biochem. 83, 677-680 (1978)). The enzyme reaction, however, was stopped by heat treatment at 95° C. for 5 minutes.

That is, one liter of hexane was added to 100 g of rice bran of Oryza sativa variety "Koshihikari" and the mixture was stirred overnight to defat the rice bran. Then 10 g of POLYCRAL AT (trademark: polyvinylpyrrolidone commercially available from GAF Chemical) and 500 ml of 10 mM Tris-HCl buffer (pH 7) containing 1 mM CaCl₂ and 5 mM 2-mercaptoethanol were added and the resulting mixture was stirred for one hour to extract enzyme. The extract was filtered through 8-ply gauze and the filtrate was centrifuged at 15,000×g for 20 minutes. The intermediate layer was collected as a crude extract. The crude extract was treated with ammonium sulfate (65% saturation) and formed precipitates were collected by centrifugation (15,000×g, 20 minutes). The precipitates were dissolved and dialyzed against the above-described buffer. After the dialysis, precipitates were removed by centrifugation to obtain a ammonium sulfate fraction.

The ammonium sulfate fraction was applied to a DEAE-Cellulose (commercially available from Whattman) column (2.0×10 cm) equilibrated with buffer A (10 mM Tris-HCl pH 7, 1 mM CaCl₂, 1 mM 2-mercaptoethanol). After washing the column with about 100 ml of buffer A containing 50 mM having linear gradient of NaCl from 50 to 350 mM. PLD was eluted in the vicinity of 0.2M of NaCl. The fractions exhibiting PLD activity were collected as an eluted solution (DEAE-Cellulose).

To the eluted solution (DEAE-Cellulose), 3M of ammonium sulfate was added to a concentration of 1M and the resultant was applied to a Phenyl Sepharose column (commercially available from PHARMACIA, 2.6×10 cm). Then the column was subjected to elution with 240 ml of buffer A having a gradient of ammonium sulfate from 1.0 to 0M. PLD was eluted in the vicinity of 0.1M of ammonium sulfate. Fractions exhibiting PLD activity were collected and dialyzed against buffer A to obtain an eluted solution (Phenyl Sepharose).

The eluted solution (Phenyl Sepharose) was applied to a Mono Q column (anion-exchange column, commercially available from PHARMACIA, 16×10 cm) and then elution was carried out with 150 ml of buffer A having a gradient of NaCl from 50-350 mM. PLD was eluted at NaCl concentrations from 210 mM to 235 mM. The fractions exhibiting PLD activity were collected and dialyzed against buffer A to obtain an eluted solution (Mono Q 1st).

The eluted solution (Mono Q 1st) was concentrated to 0.5 ml by centrifugal ultrafiltration and the resultant was applied to Superose 6 column (commercially available from PHARMACIA, 1.0-30 cm) equilibrated with buffer A containing 0.1M NaCl, and the column was subjected to elution with the same buffer. The molecular weight of the PLD was estimated as 78 kDa. The fractions exhibiting PLD activity were collected as an eluted solution (Superose 6).

To the eluted solution (Superose 6), 2.5 ml of 40% CARRIER AMPHOLITE (commercially available from PHARMACIA, pH 4.0-6.0) and distilled water were added to a final volume of 50 ml. The resultant was then subjected to isoelectric focusing using Rotofore (commercially available from BIORAD). The electrophoresis was carried out at 2° C. under a constant power of 12W for 4 hours. PLD activity was observed in the vicinity of pH4.9. The fractions exhibiting PLD activity were collected and the obtained solution was dialyzed against buffer A to obtain an isoelectric focusing fraction.

The isoelectric focusing fraction was applied to a Mono Q column (commercially available from PHARMACIA, 0.5×5 cm) and elution was carried out using buffer A having a linear gradient of NaCl from 50 mM to 350 mM. PLD was eluted in the vicinity of 210 mM and 235 mM NaCl. The two fractions exhibiting PLD activity were collected as eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II).

Purities of the eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II) were determined by SDS-polyacrylamide gel electrophoresis (Laemmli (1970)). After the electrophoresis, the gels were stained with COOMASSIE BRILLIANT BLUE R250. With either eluted solution, a main band was observed at the position of molecular weight of 82 kDa. A single band was observed with eluted solution (Mono Q 2nd-II).

By the above-described purification, eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II) were 380-fold and 760-fold purified, respectively, based on the crude extract.

The two fractions were analyzed for properties of the enzymes contained therein. The results are shown in Table 1. The buffer solutions used for measuring the optimum pH were sodium acetate (pH4-6), MES-NaOH (pH5.5-7.0) and Tris-HCl (pH7-9), all of which had a concentration of 100 mM. The pH stability means the range in which decrease in activity was not observed after leaving the enzyme to stand at 25° C. for 30 minutes. The temperature stability was measured by measuring remaining activity after leaving the enzyme to stand at 4° C., 25° C., 37° C. or 50° C. for 30 minutes. The substrate specificity was measured at a substrate concentration of 5 mM and is expressed as a relative activity taking the enzyme activity to phosphatidylcholine as 100.

                  TABLE 1                                                          ______________________________________                                                       Mono Q 2nd-I                                                                            Mono Q 2nd-II                                           ______________________________________                                         Km Value        0.29 mM    0.29 mM                                             Optimum pH      6          6                                                   pH Stability    7-8        7-8                                                 Temperature Stability                                                                          4-37° C.                                                                           4-37° C.                                     Ca.sup.2+  Dependency                                                                          20 mM or more                                                                             20 mM or more                                       Substrate Specificity                                                          Phosphatidylcholine                                                                            100        100                                                 Lysophosphatidylcholine                                                                        13         12                                                  Sphingomyelin   6          4                                                   ______________________________________                                    

2. Proof that Purified Protein is PLD

In the same manner as in the determination of the purity, eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II) were separately subjected to SDS-polyacrylamide gel electrophoresis and each of the gels was transcribed to a PVDF membrane (commercially available from MILLIPORE), followed by staining the membrane. The band corresponding to the protein having a molecular weight of 82 kDa was cut out and amino acid sequence of the N-terminal region of the protein was determined. Amino acid sequence up to 10th amino acid residue was able to be determined for both proteins, and both proteins had the same sequence as follows:

Val Gly Lys Gly Ala Thr Lys Val Tyr Ser

Although the relationship between the proteins of 82 kDa which existed in two fractions having the enzyme activity was unknown, at least the homology of the amino acid sequences thereof was thought to be high. Thus, it was thought that there was no problem even if a mixture of these fractions was used as an antigen for producing an antibody.

A mixture of the eluted solutions (Mono Q 2nd-I and Mono Q 2nd-II) was subjected to SDS-polyacrylamide gel electrophoresis employing 7.5% acrylamide and the gel was stained with COOMASSIE BRILLIANT BLUE R250. The band containing the protein of 82 kDa was cut out and the proteins were recovered by electroelution (25 mM Tris, 192 mM glycine, 0.025% SDS, 100V, 10 hours). After removing SDS by electrodialysis (15 mM ammonium bicarbonate, 200V, 5 hours), the resultant was freeze-dried. The electroelution and electrophoresis were performed by using BIOTRAP (commercially available from SCHLEICHER & SCHUELL).

Rabbits were immunized with the protein of 82 kDa highly purified by the above-described method. Immunization was performed by administering 50 μg of the protein per time at 7 days' interval. Using blood serum before the immunization and after the third immunization, immunotitration was performed. That is, PLD solution containing 8.6×10⁻³ units of the enzyme, 0 to 50 μl of the serum before the immunization or after the third immunization, 50 μl of 250 mM Tris-HCl (pH 7.0), 5 μl of 50 mM CaCl₂, 50 μl of 0.2% TRITON X-100 (trademark) and balance of water were mixed to a final volume of 250 μl and the mixture was left to stand at room temperature for 2.5 hours. To the resulting mixture, 200 μl of Protein A SEPHAROSE (commercially available from PHARMACIA) was added and the resultant was gently shaken at room temperature for 2 hours. The resultant was centrifuged (500×g, 5 minutes) and enzyme activity of the supernatant was measured. Taking the enzyme activity when no serum was added as 100%, the enzyme activities measured when 20 μl or 50 μl of the serum before the immunization were 95% and 88%, respectively, while the enzyme activities measured when 20 μl or 50 μl of the serum after the third immunization were 75% and 30%, respectively. These results proved that the protein of 82 kDa is PLD.

3. Determination of Internal Amino Acid Sequence

The PLD protein was fragmented by fragmenting the protein in a gel (Cleveland et al., J. Biol. Chem., 252, 1102(1977)). The gel containing the PLD protein, which was cut out by the same method as described in 2 was inserted in a stacking gel well on a 15% acrylamide gel prepared for separation of peptides. Staphylococcus aureus V8 protease (commercially available from WAKO PURE CHEMICAL INDUSTRIES, LTD) in an amount of 1/10 of the PLD protein was overlaid and electrophoresis was started. The electrophoresis was interrupted when bromophenol blue reached the center of the stacking gel, and 30 minutes after, the electrophoresis was restarted. After the electrophoresis, the gel was transcribed to a PVDF membrane and the PVDF membrane was stained. Clear bands were observed at positions corresponding to molecular weights of 20, 14, 13, 11 and 10 kDa. The bands corresponding to molecular weights of 20, 14 and 13 kDa were cut out and amino acid sequences of the peptide fragments contained in the bands were determined by a protein sequencer. The sequences are as follows:

20 kDa: Asn Tyr Phe His Gly Ser Asp Val Asn ? Val Leu

? Pro Arg Asn Pro Asp Asp(Asp) ? ? Ile

14 kDa: Thr ? Asn Val Gln Leu Phe Arg Ser Ile Asp Gly

Gly Ala Ala Phe Gly Phe Pro Asp Thr Pro Glu Glu Ala Ala

Lys ? Gly Leu Val Ser Gly

13 kDa: Ile Ala Met Gly Gly Tyr Gln Phe Tyr His Leu Ala Thr Arg Gln Pro Ala Arg Gly Gln Ile His Gly Phe Arg Met Ala Leu ? Tyr Glu His Leu Gly Met Leu ? Asp Val Phe

(In the sequences, "?" means the residue which could not be identified and the amino acid residue in parenthesis is one which may be another amino acid residue with a considerable probability.)

4. Preparation of cDNA Library of Rice Immature Seeds Total RNAs were prepared from immature seeds 5 days after blossom by extracting RNAs by the SDS-phenol method and by precipitating the extract with lithium chloride. Poly(A) +RNAs were prepared using OLIGOTEX-dT30 (commercially available from TAKARA SHUZO) in accordance with the instructions by the manufacturer. For cDNA cloning, cDNA SYNTHESIS SYSTEM PLUS (commercially available from AMERSHAM) and cDNA CLONING SYSTEM λgt10 (commercially available from AMERSHAM) were used. As the cloning vector, λZAPII vector (commercially available from STRATAGENE) was used and as the host cells, XL1-Blue was used.

5. Preparation of Probes

Oligonucleotides corresponding to the amino acid sequence of PLD were synthesized by a DNA synthesizer (commercially available from APPLIED BIOSYSTEMS). The sequences thereof and the amino acid sequences corresponding thereto are described below.

20KF: 5'AAYTAYTTYCAYGG 3'

20KRI: 5'RTCRTCRTCNGGRTT 3'

(wherein R represents purine bases, that is, A or G; Y represents pyrimidine bases, that is, T or C; and N represents G, A, T or C.)

20KF is a mixture of 32 types of oligonucleotides each of which encodes the amino acid sequence of

Asn Tyr Phe His Gly

that was found in the peptide having a molecular weight of 20 kDa, and 20KR1 is a mixture of 128 types of oligonucleotides each of which encodes the amino acid sequence of

Asn Pro Asp Asp (Asp)

that was found in the same peptide.

The cDNA synthesis reaction was carried out in a mixture of 10 ng of Poly(A)+RNA, 0.3 μg of random hexamer (dN6), 10 U of RNase inhibitor (RNAGuard, commercially available from PHARMACIA), 1 mM each of dATP, dCTP, dGTP and dTTP, 1×PCR buffer (commercially available from TAKARA SHUZO), 50 mM of magnesium chloride and 100 U of reverse transcriptase (M-MuLV RTase, commercially available from BRL), the total volume of the reaction mixture being 10 μl. The reaction was carried out at 37° C. for 30 minutes and the mixture was then heated at 95° C. for 5 minutes, followed by retaining the resulting mixture in ice.

Polymerase chain reaction (PCR) was performed using the above-described cDNA as a template, and 20KF and 20KR1 as primers. The reaction was carried out using 10 μl of the cDNA synthesis reaction mixture, a mixture of 50 pmol each of the primers, 200 μM each of dATP, dCTP, dGTP and dTTP, 1×PCR buffer (commercially available from TAKARA SHUZO) and 2.5 U of AmpliTaq DNA polymerase (commercially available from TAKARA SHUZO), the total volume of the reaction mixture being 50 μl. A cycle of 94° C. for 1 minute/40° C. for 1 minute/72° C. for 2.5 minutes was repeated 30 times in DNA THERMOCYCLER (commercially available from PERKIN ELMER CETUS).

PCR product was separated on 2% agarose gel. Several fragments were detected by staining the gel with ethidium bromide. One of them had a size of 94 bp which is the expected size.

The PCR fragment was cut out from the gel and subcloned into pUC19 plasmid. The subcloned PCR fragment was sequenced by the dideoxy method using T7 sequencing kit (commercially available from PHARMACIA). Between the two primers, a nucleotide sequence encoding the expected amino acid sequence was observed. The nucleotide sequence between the two primers and the amino acid sequence encoded thereby are as follows:

C TCT GAC GTG AAC TGT GTT CTA TGC CCT CGC

Ser Asp Val Asn Cys Val Leu Cys Pro Arg

Isotope ³² P (commercially available from AMERSHAM) was incorporated into the above-described oligonucleotide using DNA 5'-end labelling kit MEGALABEL (commercially available from TAKARA SHUZO) to obtain a radioactive oligonucleotide probe.

6. Screening of PLD Gene-containing Clone

Using the above-described radioactive oligonucleotide as a probe, a cDNA library was screened. The hybridization solution was 0.5M sodium phosphate buffer (pH 7.2) containing 7% SDS, 1 mM EDTA and 100 μg/ml of salmon sperm DNA, and hybridization was carried out at 45° C. for 16 hours after adding the probe to this solution. The washing solution contained 0.3M NaCl and 30 mM sodium citrate and washing was performed twice at 45° C. for 20 minutes. Positive plaques were isolated and subcloned in vivo into pBluescript plasmid (commercially available from STRATAGENE) in accordance with the instruction of the manufacturer of λZAPII cloning vector. The nucleotide sequence was determined and the region encoding the internal amino acid sequence determined in 3 existed.

7. Sequencing of 5'-end Region

Since a clone containing the full-length cDNA could not be isolated, a DNA fragment containing the 5'-end region was prepared by RACE method (Edwards et al., Nucleic Acids Res., 19, 5227-5232(1991)). 5'-AmpliFINDER RACE Kit (commercially available from CLONETECH) was used in accordance with the instructions attached to the product. OligoDNAs were prepared and PCR was performed using the mRNA prepared by the method described in 4 as a template. The PCR product was subcloned into PCRII vector (commercially available from INVITROGEN) and sequenced by the dideoxy method. As a result, it was estimated that translation is initiated from the 182th nucleotide shown in SEQ ID NO. 1 because a termination codon exists upstream thereof by 36 bp.

8. Preparation of cDNA Clone Encoding PLD Originated from Maize

Maize cDNA clone was obtained by the method described below using the DNA encoding rice PLD as a probe.

Using suspended cultured cells established by culturing a callus derived from immature embryo of maize inbred Mo 17(commercially available from MIKE BRAYTON SEEDS, INC.) in a liquid culture medium, a cDNA library was prepared by the method described in 4. However, λgt10 vector (commercially available from AMERSHAM) was used as the cloning vector and NM-514 (commercially available from AMERSHAM) was used as the host cells. Using the cDNA of rice PLD as a probe, positive plaques were isolated by the method described in 6. Phage DNA was prepared in accordance with the instructions by the manufacturer of the cloning vector. The phage DNA was digested with a restriction enzyme Kpn I and the resultant was subcloned into pBluescript plasmid. The nucleotide sequence was determined by the dideoxy method as described in 6.

9. Isolation of PLD Genome Clone Corresponding to PLD cDNA and Identification of Promoter Region

To isolate a genomic DNA clone carrying the regulatory sequence of the PLD gene corresponding to the PLD cDNA sequenced in 6, which was cloned into pBluescript plasmid, a genomic library of rice Koshihikari was prepared. This was carried out by partially digesting DNAs from leaves of Koshihikari with Mbo I, purifying fractions having sizes of 16-20 kb by sucrose gradient centrifugation, and by using lambda DASH II (commercially available from STRATAGENE) and GigapackII Gold (commercially available from STRATAGENE). Using the PLD cDNA clone as a probe, the genome library was screened. The screening was performed as described in 6. However, the hybridization was performed at 65° C. for 16 hours, the washing solution was 0.5×SSC containing 0.1% SDS and washing was performed twice at 65° C. for 20 minutes. The nucleotide sequence of the hybridized genome clone was determined by the dideoxy method. As a result, a region homologous to the cDNA sequence determined in 6 existed.

The transcription initiation site was determined by the method described in 7. In the vicinity of the transcription initiation site, "TATA" consensus sequence box was observed. The ATG translation initiation site was determined as the upstream most ATG codon in the translation reading frame of the clone and as the ATG codon which is first accessible in the mRNA synthesized in rice.

A part of the DNA sequence of the genome clone hybridized with the cDNA clone is shown in SEQ ID NO. 5. In the genome DNA sequence, a reading frame starting from the ATG translation initiation codon, which overlaps with the corresponding cDNA sequence, was identified. The promoter region is located upstream of the ATG translation initiation codon and starts immediately upstream thereof.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 7                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3040 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA to mRNA                                               (iii) HYPOTHETICAL: NO                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 182..2617                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        AGTCTCTCTTCTCCCGCAATTTTATAATCTCGATCGATCCAATCTGCTCCCCTTCTTCTT60                 CTACTCTCCCCATCTCGGCTCTCGCCATCGCCATCCTCCTCTCCCTTCCCGGAGAAGACG120                CCTCCCTCCGCCGATCACCACCCGGTAGGGCGAGGAGGGAGCCAAATCCAAATCAGCAGC180                CATGGCGCAGATGCTGCTCCATGGGACGCTGCACGCCACCATCTTC226                              MetAlaGlnMetLeuLeuHisGlyThrLeuHisAlaThrIlePhe                                  151015                                                                         GAGGCGGCGTCGCTCTCCAACCCGCACCGCGCCAGCGGAAGCGCCCCC274                            GluAlaAlaSerLeuSerAsnProHisArgAlaSerGlySerAlaPro                               202530                                                                         AAGTTCATCCGCAAGTTTGTGGAGGGGATTGAGGACACTGTGGGTGTC322                            LysPheIleArgLysPheValGluGlyIleGluAspThrValGlyVal                               354045                                                                         GGCAAAGGCGCCACCAAGGTGTATTCTACCATTGATCTGGAGAAAGCT370                            GlyLysGlyAlaThrLysValTyrSerThrIleAspLeuGluLysAla                               505560                                                                         CGTGTAGGGCGAACTAGGATGATAACCAATGAGCCCATCAACCCTCGC418                            ArgValGlyArgThrArgMetIleThrAsnGluProIleAsnProArg                               657075                                                                         TGGTATGAGTCGTTCCACATCTATTGCGCTCATATGGCTTCCAATGTG466                            TrpTyrGluSerPheHisIleTyrCysAlaHisMetAlaSerAsnVal                               80859095                                                                       ATCTTCACTGTCAAGATTGATAACCCTATTGGGGCAACGAATATTGGG514                            IlePheThrValLysIleAspAsnProIleGlyAlaThrAsnIleGly                               100105110                                                                      AGGGCTTACCTGCCTGTCCAAGAGCTTCTCAATGGAGAGGAGATTGAC562                            ArgAlaTyrLeuProValGlnGluLeuLeuAsnGlyGluGluIleAsp                               115120125                                                                      AGATGGCTCGATATCTGTGATAATAACCGCGAGTCTGTTGGTGAGAGC610                            ArgTrpLeuAspIleCysAspAsnAsnArgGluSerValGlyGluSer                               130135140                                                                      AAGATCCATGTGAAGCTTCAGTACTTCGATGTTTCCAAGGATCGCAAT658                            LysIleHisValLysLeuGlnTyrPheAspValSerLysAspArgAsn                               145150155                                                                      TGGGCGAGGGGTGTCCGCAGTACCAAGTATCCAGGTGTTCCTTACACC706                            TrpAlaArgGlyValArgSerThrLysTyrProGlyValProTyrThr                               160165170175                                                                   TTCTTCTCTCAGAGGCAAGGGTGCAAAGTTACCTTGTACCAAGATGCT754                            PhePheSerGlnArgGlnGlyCysLysValThrLeuTyrGlnAspAla                               180185190                                                                      CATGTCCCAGACAACTTCATTCCAAAGATTCCGCTTGCCGATGGCAAG802                            HisValProAspAsnPheIleProLysIleProLeuAlaAspGlyLys                               195200205                                                                      AATTATGAACCCCACAGATGCTGGGAGGATATCTTTGATGCTATAAGC850                            AsnTyrGluProHisArgCysTrpGluAspIlePheAspAlaIleSer                               210215220                                                                      AATGCTCAACATTTGATTTACATCACTGGCTGGTCTGTATACACTGAG898                            AsnAlaGlnHisLeuIleTyrIleThrGlyTrpSerValTyrThrGlu                               225230235                                                                      ATCACCTTGGTTAGGGACTCCAATCGTCCAAAACCTGGAGGGGATGTC946                            IleThrLeuValArgAspSerAsnArgProLysProGlyGlyAspVal                               240245250255                                                                   ACCCTTGGGGAGTTGCTCAAGAAGAAGGCCAGTGAAGGTGTTCGGGTC994                            ThrLeuGlyGluLeuLeuLysLysLysAlaSerGluGlyValArgVal                               260265270                                                                      CTCATGCTTGTGTGGGATGACAGGACTTCAGTTGGTTTGCTAAAGAGG1042                           LeuMetLeuValTrpAspAspArgThrSerValGlyLeuLeuLysArg                               275280285                                                                      GATGGCTTGATGGCAACACATGATGAGGAAACTGAAAATTACTTCCAT1090                           AspGlyLeuMetAlaThrHisAspGluGluThrGluAsnTyrPheHis                               290295300                                                                      GGCTCTGACGTGAACTGTGTTCTATGCCCTCGCAACCCTGATGACTCA1138                           GlySerAspValAsnCysValLeuCysProArgAsnProAspAspSer                               305310315                                                                      GGCAGCATTGTTCAGGATCTGTCGATCTCAACTATGTTTACACACCAT1186                           GlySerIleValGlnAspLeuSerIleSerThrMetPheThrHisHis                               320325330335                                                                   CAGAAGATAGTAGTTGTTGACCATGAGTTGCCAAACCAGGGCTCCCAA1234                           GlnLysIleValValValAspHisGluLeuProAsnGlnGlySerGln                               340345350                                                                      CAAAGGAGGATAGTCAGTTTCGTTGGTGGCCTTGATCTCTGTGATGGA1282                           GlnArgArgIleValSerPheValGlyGlyLeuAspLeuCysAspGly                               355360365                                                                      AGGTATGACACTCAGTACCATTCTTTGTTTAGGACACTCGACAGTACC1330                           ArgTyrAspThrGlnTyrHisSerLeuPheArgThrLeuAspSerThr                               370375380                                                                      CATCATGATGACTTCCACCAGCCAAACTTTGCCACTGCATCAATCAAA1378                           HisHisAspAspPheHisGlnProAsnPheAlaThrAlaSerIleLys                               385390395                                                                      AAGGGTGGACCTAGAGAGCCATGGCATGATATTCACTCACGGCTGGAA1426                           LysGlyGlyProArgGluProTrpHisAspIleHisSerArgLeuGlu                               400405410415                                                                   GGGCCAATCGCATGGGATGTTCTTTACAATTTCGAGCAGAGATGGAGA1474                           GlyProIleAlaTrpAspValLeuTyrAsnPheGluGlnArgTrpArg                               420425430                                                                      AAGCAGGGTGGTAAGGATCTCCTTCTGCAGCTCAGGGATCTGTCTGAC1522                           LysGlnGlyGlyLysAspLeuLeuLeuGlnLeuArgAspLeuSerAsp                               435440445                                                                      ACTATTATTCCACCTTCTCCTGTTATGTTTCCAGAGGACAGAGAAACA1570                           ThrIleIleProProSerProValMetPheProGluAspArgGluThr                               450455460                                                                      TGGAATGTTCAGCTATTTAGATCCATTGATGGTGGTGCTGCTTTTGGG1618                           TrpAsnValGlnLeuPheArgSerIleAspGlyGlyAlaAlaPheGly                               465470475                                                                      TTCCCTGATACCCCTGAGGAGGCTGCAAAAGCTGGGCTTGTAAGCGGA1666                           PheProAspThrProGluGluAlaAlaLysAlaGlyLeuValSerGly                               480485490495                                                                   AAGGATCAAATCATTGACAGGAGCATCCAGGATGCATACATACATGCC1714                           LysAspGlnIleIleAspArgSerIleGlnAspAlaTyrIleHisAla                               500505510                                                                      ATCCGGAGGGCAAAGAACTTCATCTATATAGAGAACCAATACTTCCTT1762                           IleArgArgAlaLysAsnPheIleTyrIleGluAsnGlnTyrPheLeu                               515520525                                                                      GGAAGTTCCTATGCCTGGAAACCCGAGGGCATCAAGCCTGAAGACATT1810                           GlySerSerTyrAlaTrpLysProGluGlyIleLysProGluAspIle                               530535540                                                                      GGTGCCCTGCATTTGATTCCTAAGGAGCTTGCACTGAAAGTTGTCAGT1858                           GlyAlaLeuHisLeuIleProLysGluLeuAlaLeuLysValValSer                               545550555                                                                      AAGATTGAAGCCGGGGAACGGTTCACTGTTTATGTTGTGGTGCCAATG1906                           LysIleGluAlaGlyGluArgPheThrValTyrValValValProMet                               560565570575                                                                   TGGCCTGAGGGTGTTCCAGAGAGTGGATCTGTTCAGGCAATCCTGGAC1954                           TrpProGluGlyValProGluSerGlySerValGlnAlaIleLeuAsp                               580585590                                                                      TGGCAAAGGAGAACAATGGAGATGATGTACACTGACATTACAGAGGCT2002                           TrpGlnArgArgThrMetGluMetMetTyrThrAspIleThrGluAla                               595600605                                                                      CTCCAAGCCAAGGGAATTGAAGCGAACCCCAAGGACTACCTCACTTTC2050                           LeuGlnAlaLysGlyIleGluAlaAsnProLysAspTyrLeuThrPhe                               610615620                                                                      TTCTGCTTGGGTAACCGTGAGGTGAAGCAGGCTGGGGAATATCAGCCT2098                           PheCysLeuGlyAsnArgGluValLysGlnAlaGlyGluTyrGlnPro                               625630635                                                                      GAAGAACAACCAGAAGCTGACACTGATTACAGCCGAGCTCAGGAAGCT2146                           GluGluGlnProGluAlaAspThrAspTyrSerArgAlaGlnGluAla                               640645650655                                                                   AGGAGGTTCATGATCTATGTCCACACCAAAATGATGATAGTTGACGAT2194                           ArgArgPheMetIleTyrValHisThrLysMetMetIleValAspAsp                               660665670                                                                      GAGTACATCATCATCGGTTCTGCAAACATCAACCAGAGGTCGATGGAC2242                           GluTyrIleIleIleGlySerAlaAsnIleAsnGlnArgSerMetAsp                               675680685                                                                      GGCGCTAGGGACTCTGAGATCGCCATGGGCGGGTACCAGCCATACCAT2290                           GlyAlaArgAspSerGluIleAlaMetGlyGlyTyrGlnProTyrHis                               690695700                                                                      CTGGCGACCAGGCAACCAGCCCGTGGCCAGATCCATGGCTTCCGGATG2338                           LeuAlaThrArgGlnProAlaArgGlyGlnIleHisGlyPheArgMet                               705710715                                                                      GCGCTGTGGTACGAGCACCTGGGAATGCTGGATGATGTGTTCCAGCGC2386                           AlaLeuTrpTyrGluHisLeuGlyMetLeuAspAspValPheGlnArg                               720725730735                                                                   CCCGAGAGCCTGGAGTGTGTGCAGAAGGTGAACAGGATCGCGGAGAAG2434                           ProGluSerLeuGluCysValGlnLysValAsnArgIleAlaGluLys                               740745750                                                                      TACTGGGACATGTACTCCAGCGACGACCTCCAGCAGGACCTCCCTGGC2482                           TyrTrpAspMetTyrSerSerAspAspLeuGlnGlnAspLeuProGly                               755760765                                                                      CACCTCCTCAGCTACCCCATTGGCGTCGCCAGCGATGGTGTGGTGACT2530                           HisLeuLeuSerTyrProIleGlyValAlaSerAspGlyValValThr                               770775780                                                                      GAGCTGCCCGGGATGGAGTACTTTCCTGACACACGGGCCCGCGTCCTC2578                           GluLeuProGlyMetGluTyrPheProAspThrArgAlaArgValLeu                               785790795                                                                      GGCGCCAAGTCGGATTACATGCCCCCCATCCTCACCTCATAGACGAGGA2627                          GlyAlaLysSerAspTyrMetProProIleLeuThrSer                                        800805810                                                                      AGCACTACACTACAATCTGCTGGCTTCTCCTGTCAGTCCTTCTGTACTTCTTCAGTTTGG2687               TGGCGAGATGGTATGGCCGTTGTTCAGAATTTCTTCAGAATAGCAGTTGTTACAGTTGTG2747               AATCATAAAGTAATAAGTGCAGTATCTGTGCATGGTTGAGTTGGGAAGAAGATCGGGGAT2807               GCAATGATGCTTGTGAAGTTGTGATGCCGTTTGTAAGATGGGAAGTTGGGAACTACTAAG2867               TAATTGGCATGATTGTACTTTGCACTACTGTTTAGCGTTGTTGATACTGGTTAACCGTGT2927               GTTCATCTGAACTTGATTCTTGATGCAGTTTGTGGCATTACCAGTTTATCATCGTTCTTC2987               AGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3040                      (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 812 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetAlaGlnMetLeuLeuHisGlyThrLeuHisAlaThrIlePheGlu                               151015                                                                         AlaAlaSerLeuSerAsnProHisArgAlaSerGlySerAlaProLys                               202530                                                                         PheIleArgLysPheValGluGlyIleGluAspThrValGlyValGly                               354045                                                                         LysGlyAlaThrLysValTyrSerThrIleAspLeuGluLysAlaArg                               505560                                                                         ValGlyArgThrArgMetIleThrAsnGluProIleAsnProArgTrp                               65707580                                                                       TyrGluSerPheHisIleTyrCysAlaHisMetAlaSerAsnValIle                               859095                                                                         PheThrValLysIleAspAsnProIleGlyAlaThrAsnIleGlyArg                               100105110                                                                      AlaTyrLeuProValGlnGluLeuLeuAsnGlyGluGluIleAspArg                               115120125                                                                      TrpLeuAspIleCysAspAsnAsnArgGluSerValGlyGluSerLys                               130135140                                                                      IleHisValLysLeuGlnTyrPheAspValSerLysAspArgAsnTrp                               145150155160                                                                   AlaArgGlyValArgSerThrLysTyrProGlyValProTyrThrPhe                               165170175                                                                      PheSerGlnArgGlnGlyCysLysValThrLeuTyrGlnAspAlaHis                               180185190                                                                      ValProAspAsnPheIleProLysIleProLeuAlaAspGlyLysAsn                               195200205                                                                      TyrGluProHisArgCysTrpGluAspIlePheAspAlaIleSerAsn                               210215220                                                                      AlaGlnHisLeuIleTyrIleThrGlyTrpSerValTyrThrGluIle                               225230235240                                                                   ThrLeuValArgAspSerAsnArgProLysProGlyGlyAspValThr                               245250255                                                                      LeuGlyGluLeuLeuLysLysLysAlaSerGluGlyValArgValLeu                               260265270                                                                      MetLeuValTrpAspAspArgThrSerValGlyLeuLeuLysArgAsp                               275280285                                                                      GlyLeuMetAlaThrHisAspGluGluThrGluAsnTyrPheHisGly                               290295300                                                                      SerAspValAsnCysValLeuCysProArgAsnProAspAspSerGly                               305310315320                                                                   SerIleValGlnAspLeuSerIleSerThrMetPheThrHisHisGln                               325330335                                                                      LysIleValValValAspHisGluLeuProAsnGlnGlySerGlnGln                               340345350                                                                      ArgArgIleValSerPheValGlyGlyLeuAspLeuCysAspGlyArg                               355360365                                                                      TyrAspThrGlnTyrHisSerLeuPheArgThrLeuAspSerThrHis                               370375380                                                                      HisAspAspPheHisGlnProAsnPheAlaThrAlaSerIleLysLys                               385390395400                                                                   GlyGlyProArgGluProTrpHisAspIleHisSerArgLeuGluGly                               405410415                                                                      ProIleAlaTrpAspValLeuTyrAsnPheGluGlnArgTrpArgLys                               420425430                                                                      GlnGlyGlyLysAspLeuLeuLeuGlnLeuArgAspLeuSerAspThr                               435440445                                                                      IleIleProProSerProValMetPheProGluAspArgGluThrTrp                               450455460                                                                      AsnValGlnLeuPheArgSerIleAspGlyGlyAlaAlaPheGlyPhe                               465470475480                                                                   ProAspThrProGluGluAlaAlaLysAlaGlyLeuValSerGlyLys                               485490495                                                                      AspGlnIleIleAspArgSerIleGlnAspAlaTyrIleHisAlaIle                               500505510                                                                      ArgArgAlaLysAsnPheIleTyrIleGluAsnGlnTyrPheLeuGly                               515520525                                                                      SerSerTyrAlaTrpLysProGluGlyIleLysProGluAspIleGly                               530535540                                                                      AlaLeuHisLeuIleProLysGluLeuAlaLeuLysValValSerLys                               545550555560                                                                   IleGluAlaGlyGluArgPheThrValTyrValValValProMetTrp                               565570575                                                                      ProGluGlyValProGluSerGlySerValGlnAlaIleLeuAspTrp                               580585590                                                                      GlnArgArgThrMetGluMetMetTyrThrAspIleThrGluAlaLeu                               595600605                                                                      GlnAlaLysGlyIleGluAlaAsnProLysAspTyrLeuThrPhePhe                               610615620                                                                      CysLeuGlyAsnArgGluValLysGlnAlaGlyGluTyrGlnProGlu                               625630635640                                                                   GluGlnProGluAlaAspThrAspTyrSerArgAlaGlnGluAlaArg                               645650655                                                                      ArgPheMetIleTyrValHisThrLysMetMetIleValAspAspGlu                               660665670                                                                      TyrIleIleIleGlySerAlaAsnIleAsnGlnArgSerMetAspGly                               675680685                                                                      AlaArgAspSerGluIleAlaMetGlyGlyTyrGlnProTyrHisLeu                               690695700                                                                      AlaThrArgGlnProAlaArgGlyGlnIleHisGlyPheArgMetAla                               705710715720                                                                   LeuTrpTyrGluHisLeuGlyMetLeuAspAspValPheGlnArgPro                               725730735                                                                      GluSerLeuGluCysValGlnLysValAsnArgIleAlaGluLysTyr                               740745750                                                                      TrpAspMetTyrSerSerAspAspLeuGlnGlnAspLeuProGlyHis                               755760765                                                                      LeuLeuSerTyrProIleGlyValAlaSerAspGlyValValThrGlu                               770775780                                                                      LeuProGlyMetGluTyrPheProAspThrArgAlaArgValLeuGly                               785790795800                                                                   AlaLysSerAspTyrMetProProIleLeuThrSer                                           805810                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2804 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA to mRNA                                               (iii) HYPOTHETICAL: NO                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 107..2542                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        ATCACCGTCGTCATCAATCACGGTGACCTTGCCCTCGCTGCCCGACTGGAACCGGACGCT60                 GCTGCTGCTGGTAGGCTGACAGCGAGGAGGACGAGACGAGGGGGCCATGGCTCAG115                     MetAlaGln                                                                      815                                                                            ATCTTGCTCCACGGCACGCTCCACGCCACCATCTTCGAGGCCGAGTCG163                            IleLeuLeuHisGlyThrLeuHisAlaThrIlePheGluAlaGluSer                               820825830                                                                      CTCTCCAACCCGCACCGCGCCACTGGCGGCGCCCCCAAGTTCATCCGC211                            LeuSerAsnProHisArgAlaThrGlyGlyAlaProLysPheIleArg                               835840845                                                                      AAGCTTGTGGAAGGGATCGAGGACACCGTGGGTGTCGGCAAGGGCGCC259                            LysLeuValGluGlyIleGluAspThrValGlyValGlyLysGlyAla                               850855860                                                                      ACCAAGATATATGCCACCGTCGATCTCGAGAAGGCCCGTGTCGGGCGG307                            ThrLysIleTyrAlaThrValAspLeuGluLysAlaArgValGlyArg                               865870875                                                                      ACCCGGATGATCTCCAACGAGCCCGTGAACCCTCGTTGGTACGAGTCC355                            ThrArgMetIleSerAsnGluProValAsnProArgTrpTyrGluSer                               880885890895                                                                   TTCCACATCTACTGCGCGCACATGGCCGCCGACGTCATCTTCACCGTC403                            PheHisIleTyrCysAlaHisMetAlaAlaAspValIlePheThrVal                               900905910                                                                      AAGATCGACAACTCCATCGGGGCCTCGCTCATCGGGAGGGCCTACTTG451                            LysIleAspAsnSerIleGlyAlaSerLeuIleGlyArgAlaTyrLeu                               915920925                                                                      GCTGTCCAGGACCTCCTGGGAGGGGAGGAGATCGACAAGTGGCTTGAA499                            AlaValGlnAspLeuLeuGlyGlyGluGluIleAspLysTrpLeuGlu                               930935940                                                                      ATCTCCGATGAAAATCGTGAGCCTGTTGGGGACAGCAAGATCCATGTG547                            IleSerAspGluAsnArgGluProValGlyAspSerLysIleHisVal                               945950955                                                                      AAGCTCCAGTACTTTGACGTCGGCAAGGACCGTAACTGGGCGAGGGGT595                            LysLeuGlnTyrPheAspValGlyLysAspArgAsnTrpAlaArgGly                               960965970975                                                                   GTCCGGAGCACCAAGTACCCTGGTGTCCCTTACACCTTCTTCTCGCAG643                            ValArgSerThrLysTyrProGlyValProTyrThrPhePheSerGln                               980985990                                                                      AGGCAGGGGTGTAAGGTTACTCTGTACCAGGACGCTCATGTGCCGGAC691                            ArgGlnGlyCysLysValThrLeuTyrGlnAspAlaHisValProAsp                               99510001005                                                                    AACTTTGTTCCCAGGATCCAGCTCGCTGATGGCAAGAACTATGAGCCG739                            AsnPheValProArgIleGlnLeuAlaAspGlyLysAsnTyrGluPro                               101010151020                                                                   CACAGGTGCTGGGAGGATATCTTTGATGCTATAAGCAAGGCTCAGCAT787                            HisArgCysTrpGluAspIlePheAspAlaIleSerLysAlaGlnHis                               102510301035                                                                   TTGATTTACATCACTGGCTGGTCCGTGTACACAGAGATCACCTTGGTC835                            LeuIleTyrIleThrGlyTrpSerValTyrThrGluIleThrLeuVal                               1040104510501055                                                               AGGGACACCAACAGGCCAAAACCTGGTGGTGATGTTACTCTTGGGGAG883                            ArgAspThrAsnArgProLysProGlyGlyAspValThrLeuGlyGlu                               106010651070                                                                   TTGCTCAAGAGGAAGGCCAGTGAAGGTGTCCGGGTGCTTATGCTGGTG931                            LeuLeuLysArgLysAlaSerGluGlyValArgValLeuMetLeuVal                               107510801085                                                                   TGGGATGACAGGACTTCTGTCGGCCTGCTTAAGAAGGATGGCTTGATG979                            TrpAspAspArgThrSerValGlyLeuLeuLysLysAspGlyLeuMet                               109010951100                                                                   GCTACCCATGATGAGGAGACTGCAAATTACTTCCATGGCACGGATGTC1027                           AlaThrHisAspGluGluThrAlaAsnTyrPheHisGlyThrAspVal                               110511101115                                                                   AACTGTGTTCTGTGCCCTCGCAACCCTGATGATTCTGGCAGCTTTGTC1075                           AsnCysValLeuCysProArgAsnProAspAspSerGlySerPheVal                               1120112511301135                                                               CAGGATCTGCAGATATCAACTATGTTCACGCACCACCAGAAGATAGTA1123                           GlnAspLeuGlnIleSerThrMetPheThrHisHisGlnLysIleVal                               114011451150                                                                   GTAGTCGACCATGAGATGCCGAACCAGGGATCCCAGCAAAGGAGGATA1171                           ValValAspHisGluMetProAsnGlnGlySerGlnGlnArgArgIle                               115511601165                                                                   GTCAGCTTCATTGGTGGCATTGACCTTTGTGATGGAAGATATGATACC1219                           ValSerPheIleGlyGlyIleAspLeuCysAspGlyArgTyrAspThr                               117011751180                                                                   CAGTACCACTCCTTGTTCAGGACGCTTGACACTGTCCATCACGATGAC1267                           GlnTyrHisSerLeuPheArgThrLeuAspThrValHisHisAspAsp                               118511901195                                                                   TTCCACCAGCCGAACTTTGAGGGTGGGTCAATCAAGAAAGGTGGCCCA1315                           PheHisGlnProAsnPheGluGlyGlySerIleLysLysGlyGlyPro                               1200120512101215                                                               AGGGAGCCATGGCATGATATCCACTCACGGTTGGAAGGGCCAATCGCT1363                           ArgGluProTrpHisAspIleHisSerArgLeuGluGlyProIleAla                               122012251230                                                                   TGGGATGTTCTTTACAACTTTGAGCAGAGATGGAGAAAGCAGGGTGGT1411                           TrpAspValLeuTyrAsnPheGluGlnArgTrpArgLysGlnGlyGly                               123512401245                                                                   AAGGACCTCCTTGTGCGTCTCAGGGATCTTCCTGACATTATCATCCCC1459                           LysAspLeuLeuValArgLeuArgAspLeuProAspIleIleIlePro                               125012551260                                                                   CCTTCTCCTGTGATGTTCCCGGAGGACAGAGAGACATGGAATGTTCAG1507                           ProSerProValMetPheProGluAspArgGluThrTrpAsnValGln                               126512701275                                                                   CTCTTCAGATCCATCGATGGTGGTGCTGCTTTTGGCTTCCCCGAGACT1555                           LeuPheArgSerIleAspGlyGlyAlaAlaPheGlyPheProGluThr                               1280128512901295                                                               CCCGAGGAAGCTGCAAGAGCTGGGCTTGTGAGTGGAAAGGATCAAATC1603                           ProGluGluAlaAlaArgAlaGlyLeuValSerGlyLysAspGlnIle                               130013051310                                                                   ATCGACCGGAGTATCCAGGATGCATACGTAAACGCCATACGGAGGGCG1651                           IleAspArgSerIleGlnAspAlaTyrValAsnAlaIleArgArgAla                               131513201325                                                                   AAGAACTTCATCTACATTGAGAATCAGTACTTCCTTGGAAGTTCATAC1699                           LysAsnPheIleTyrIleGluAsnGlnTyrPheLeuGlySerSerTyr                               133013351340                                                                   GGCTGGAAGCCCGAAGGCATCAAGCCGGAAGAAATCGGTGCTCTTCAC1747                           GlyTrpLysProGluGlyIleLysProGluGluIleGlyAlaLeuHis                               134513501355                                                                   TTGATTCCGAAGGAGCTCTCGCTGAAGATTGTCAGCAAGATTGAAGCT1795                           LeuIleProLysGluLeuSerLeuLysIleValSerLysIleGluAla                               1360136513701375                                                               GGGGAGCGGTTTACTGTTTATGTTGTGGTGCCAATGTGGCCTGAGGGT1843                           GlyGluArgPheThrValTyrValValValProMetTrpProGluGly                               138013851390                                                                   GTTCCAGAAAGCGCTTCTGTTCAGGCAATCCTTGACTGGCAAAGGAGA1891                           ValProGluSerAlaSerValGlnAlaIleLeuAspTrpGlnArgArg                               139514001405                                                                   ACGATGGAGATGATGTACACTGACATCGCACAAGCTCTCGAAGCCAAC1939                           ThrMetGluMetMetTyrThrAspIleAlaGlnAlaLeuGluAlaAsn                               141014151420                                                                   GGGATTGAAGCAAACCCCAAGGACTATCTCACTTTCTTCTGCTTAGGT1987                           GlyIleGluAlaAsnProLysAspTyrLeuThrPhePheCysLeuGly                               142514301435                                                                   AACCGTGAGGTAAAGCAGGAGGGAGAATATGAACCAGAGGAGCACCCA2035                           AsnArgGluValLysGlnGluGlyGluTyrGluProGluGluHisPro                               1440144514501455                                                               GAACCTGACACTGATTACATCCGGGCTCAAGAGGCTAGGAGGTTTATG2083                           GluProAspThrAspTyrIleArgAlaGlnGluAlaArgArgPheMet                               146014651470                                                                   ATCTATGTTCATACCAAAATGATGATAGTGGACGACGAGTACATCATC2131                           IleTyrValHisThrLysMetMetIleValAspAspGluTyrIleIle                               147514801485                                                                   ATTGGGTCCGCCAACATCAACCAGAGGTCCATGGACGGTGCCAGGGAC2179                           IleGlySerAlaAsnIleAsnGlnArgSerMetAspGlyAlaArgAsp                               149014951500                                                                   TCCGAGATCGCCATGGGCGCGTACCAGCCGTACCACTTGGCGACTAGG2227                           SerGluIleAlaMetGlyAlaTyrGlnProTyrHisLeuAlaThrArg                               150515101515                                                                   CAGCCTGCCCGGGGCCAGATCCATGGCTTCCGGATGTCTCTTTGGTAC2275                           GlnProAlaArgGlyGlnIleHisGlyPheArgMetSerLeuTrpTyr                               1520152515301535                                                               GAGCACCTGGGAATGCTGGAAGACGTCTTCCAGCGGCCCGAGAGCGTA2323                           GluHisLeuGlyMetLeuGluAspValPheGlnArgProGluSerVal                               154015451550                                                                   GAGTGTGTGCAGAAGGTGAACGAGGTCGCCGAGAAGTACTGGGACCTG2371                           GluCysValGlnLysValAsnGluValAlaGluLysTyrTrpAspLeu                               155515601565                                                                   TACTCGAGCGACGACCTGGAGCAGGACCTCCCGGGCCACCTCCTCAGC2419                           TyrSerSerAspAspLeuGluGlnAspLeuProGlyHisLeuLeuSer                               157015751580                                                                   TACCCGATCGGTGTCACTGCCGACGGCAGCGTTACCGAGCTGCCCGGG2467                           TyrProIleGlyValThrAlaAspGlySerValThrGluLeuProGly                               158515901595                                                                   ATGGAGAACTTCCCCGACACCCGCGCCCGCGTCCTCGGGAACAAGTCG2515                           MetGluAsnPheProAspThrArgAlaArgValLeuGlyAsnLysSer                               1600160516101615                                                               GATTACCTCCCGCCCATCCTCACCACATAGAGTGCACACTGCAGGCA2562                            AspTyrLeuProProIleLeuThrThr                                                    1620                                                                           GCGCCATGGCTGCTCTCCTCTCTGGCCTCACCTTGGTGTCCCTGTGTTTGTGTTTGGGAC2622               ACTGGAGGTTCAGATTGCAGTGTTGATATTATATCCCCCCTCCGTCCAGAGGGATTCGAC2682               GTTATTGAGTCATAATAAAATGCATTGTGCACGGTGGGAGACTGGGAGGATAGGAATTAT2742               AGTTGTTTATTACAGTACGACTGCTTACTGCATCCAGATTGTGTTGTCCCTAAAAAAAAA2802               AA2804                                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 812 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetAlaGlnIleLeuLeuHisGlyThrLeuHisAlaThrIlePheGlu                               151015                                                                         AlaGluSerLeuSerAsnProHisArgAlaThrGlyGlyAlaProLys                               202530                                                                         PheIleArgLysLeuValGluGlyIleGluAspThrValGlyValGly                               354045                                                                         LysGlyAlaThrLysIleTyrAlaThrValAspLeuGluLysAlaArg                               505560                                                                         ValGlyArgThrArgMetIleSerAsnGluProValAsnProArgTrp                               65707580                                                                       TyrGluSerPheHisIleTyrCysAlaHisMetAlaAlaAspValIle                               859095                                                                         PheThrValLysIleAspAsnSerIleGlyAlaSerLeuIleGlyArg                               100105110                                                                      AlaTyrLeuAlaValGlnAspLeuLeuGlyGlyGluGluIleAspLys                               115120125                                                                      TrpLeuGluIleSerAspGluAsnArgGluProValGlyAspSerLys                               130135140                                                                      IleHisValLysLeuGlnTyrPheAspValGlyLysAspArgAsnTrp                               145150155160                                                                   AlaArgGlyValArgSerThrLysTyrProGlyValProTyrThrPhe                               165170175                                                                      PheSerGlnArgGlnGlyCysLysValThrLeuTyrGlnAspAlaHis                               180185190                                                                      ValProAspAsnPheValProArgIleGlnLeuAlaAspGlyLysAsn                               195200205                                                                      TyrGluProHisArgCysTrpGluAspIlePheAspAlaIleSerLys                               210215220                                                                      AlaGlnHisLeuIleTyrIleThrGlyTrpSerValTyrThrGluIle                               225230235240                                                                   ThrLeuValArgAspThrAsnArgProLysProGlyGlyAspValThr                               245250255                                                                      LeuGlyGluLeuLeuLysArgLysAlaSerGluGlyValArgValLeu                               260265270                                                                      MetLeuValTrpAspAspArgThrSerValGlyLeuLeuLysLysAsp                               275280285                                                                      GlyLeuMetAlaThrHisAspGluGluThrAlaAsnTyrPheHisGly                               290295300                                                                      ThrAspValAsnCysValLeuCysProArgAsnProAspAspSerGly                               305310315320                                                                   SerPheValGlnAspLeuGlnIleSerThrMetPheThrHisHisGln                               325330335                                                                      LysIleValValValAspHisGluMetProAsnGlnGlySerGlnGln                               340345350                                                                      ArgArgIleValSerPheIleGlyGlyIleAspLeuCysAspGlyArg                               355360365                                                                      TyrAspThrGlnTyrHisSerLeuPheArgThrLeuAspThrValHis                               370375380                                                                      HisAspAspPheHisGlnProAsnPheGluGlyGlySerIleLysLys                               385390395400                                                                   GlyGlyProArgGluProTrpHisAspIleHisSerArgLeuGluGly                               405410415                                                                      ProIleAlaTrpAspValLeuTyrAsnPheGluGlnArgTrpArgLys                               420425430                                                                      GlnGlyGlyLysAspLeuLeuValArgLeuArgAspLeuProAspIle                               435440445                                                                      IleIleProProSerProValMetPheProGluAspArgGluThrTrp                               450455460                                                                      AsnValGlnLeuPheArgSerIleAspGlyGlyAlaAlaPheGlyPhe                               465470475480                                                                   ProGluThrProGluGluAlaAlaArgAlaGlyLeuValSerGlyLys                               485490495                                                                      AspGlnIleIleAspArgSerIleGlnAspAlaTyrValAsnAlaIle                               500505510                                                                      ArgArgAlaLysAsnPheIleTyrIleGluAsnGlnTyrPheLeuGly                               515520525                                                                      SerSerTyrGlyTrpLysProGluGlyIleLysProGluGluIleGly                               530535540                                                                      AlaLeuHisLeuIleProLysGluLeuSerLeuLysIleValSerLys                               545550555560                                                                   IleGluAlaGlyGluArgPheThrValTyrValValValProMetTrp                               565570575                                                                      ProGluGlyValProGluSerAlaSerValGlnAlaIleLeuAspTrp                               580585590                                                                      GlnArgArgThrMetGluMetMetTyrThrAspIleAlaGlnAlaLeu                               595600605                                                                      GluAlaAsnGlyIleGluAlaAsnProLysAspTyrLeuThrPhePhe                               610615620                                                                      CysLeuGlyAsnArgGluValLysGlnGluGlyGluTyrGluProGlu                               625630635640                                                                   GluHisProGluProAspThrAspTyrIleArgAlaGlnGluAlaArg                               645650655                                                                      ArgPheMetIleTyrValHisThrLysMetMetIleValAspAspGlu                               660665670                                                                      TyrIleIleIleGlySerAlaAsnIleAsnGlnArgSerMetAspGly                               675680685                                                                      AlaArgAspSerGluIleAlaMetGlyAlaTyrGlnProTyrHisLeu                               690695700                                                                      AlaThrArgGlnProAlaArgGlyGlnIleHisGlyPheArgMetSer                               705710715720                                                                   LeuTrpTyrGluHisLeuGlyMetLeuGluAspValPheGlnArgPro                               725730735                                                                      GluSerValGluCysValGlnLysValAsnGluValAlaGluLysTyr                               740745750                                                                      TrpAspLeuTyrSerSerAspAspLeuGluGlnAspLeuProGlyHis                               755760765                                                                      LeuLeuSerTyrProIleGlyValThrAlaAspGlySerValThrGlu                               770775780                                                                      LeuProGlyMetGluAsnPheProAspThrArgAlaArgValLeuGly                               785790795800                                                                   AsnLysSerAspTyrLeuProProIleLeuThrThr                                           805810                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2799 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1876..1983                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 2524..2799                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        CAAGGGTGTACATAGATTTGTCTCGTAAAATAGTATTATAATATTATAAACTTATTACTC60                 TATCCGTTCTAAAATATAAGAACCTTATGACTGGATGGAACATTTCCTAGTACTACGAAT120                CTGAACACATGTCTAGATTCATAGTACTAGGAAATGTCTCATCGCGGTACTAGGTTCTTA180                TATTTTAGGATGGAGGGAGTTTAATATAAAACTAATGGTTAGAACTTTGAAAGTTTGATT240                TTAAATGTCAAATATTTATGGCTGGAGGTAGTATAATATGTTTTTTTTGGGACGTAGACT300                AGGTAGTATAATATGTTTGGTTGTGTTTAGATCCAATATTTGGATCCAAACTTCAGTCAT360                TTTCCATCACATCAACTTGTCATATACACATAACTTTTCAGTCACATCATCCCCAATTTC420                AACCAAAATCAAACTTTGCGCTGAACTAAACACAACCTTTGGGCCCGTTTAGTTCCCCAA480                TTTTTTTCCCAAAAACATCACATCGAATCTTTGGACACATGCATGAAGCATTAAATATAG540                ATAAAAAGAAAAACTAATTGCACAGTTATGGAGGAAATCGCGAGACGAATCTTTTAAGCC600                TAATTAGTCCGTGATTAGCCATAAGTGCTACAGTAACCCAATTGTGCTAATGACGGCTTA660                ATTAGTCTCCACAAGATTCGTCTCGCAGTTTCCAGGCGAGTTCTGAAATTAGTTTTTTCA720                TTCGTGTCCGAAAACCCCTTCCGACATCCGGTCAAACGTTCGATATGACACCCACAAATT780                TTCTTTTCCCCAACTAAACACACCCTTTATCTCTTACCCTCTGGCTCTTTCAGTAGGCAT840                ATCCAAGACAGCTGGTAATGCAGGCTCGGACATAATTTGACAGTTACGTTCATGTGACCG900                ACGGTTGATGCTAGTGCAACTGCAACATACTGTTCAGATGGATGTCCCAACGAGCTCAAA960                ACAACTTAGGTGGCGCGTCGCGATTCATCAATAACTCAAATGGAAGCGCAAGTGCACGTA1020               CGAAAATGACAGCGAGTGAGGTGGCGAGCCTCACCTTGGTGATCCCAACCGGATAAGCTA1080               TGCATCAGCCAGTTTCGTGGGGCTGCACATTTCGTCGAACACCTGGAGTCCACGCCGCCG1140               GCGACGTCGGCACAGCGCGCCCGCCCACCGCCCACGCACGCGCTTGACTCCACCCATGTT1200               CTCCCTTCTCGACGCCCGCGAAGCCAGCGAACCGATCCGAGGAAGTCAAGCCCCCACCGC1260               CACTTGGACCGACCTCGGGACGACGACGCCCCCGCGCTCTTCTAGACGCGCGGACGACGC1320               GGGCGCTGGCTCCGCGACGCGACGTCGCGGTCATGGAGTAACCGCGACGGACAGATACTT1380               CTACCCGTTTTTAACCTCGCCTCCTCCTCCTCCCGGCTCGAGATCCGTGGCCACGACGCG1440               TGGTGGGAAACCGGGAACGACGTGCACGCACGCACACAGGGCAAGTTTCAGTAGAAAAAT1500               CGCCGGCATCCAGATCGGGACAGTCTCTCTTCTCCCGCAATTTTATAATCTCGCTCGATC1560               CAATCTGCTCCCCTTCTTCTTCTACTCTCCCCATCTCGGCTCTCGCCATCGCCATCCTCC1620               TCTCCCTTCCCGGAGAAGACGCCTCCCTCCGCCGATCACCACCCGGTAAGCCCAGTGTGC1680               TTAGGCTAAGCGCACTAGAGCTTCTTGCTCGCTTGCTTCTTCTCCGCTCAGATCTGCTTG1740               CTTGCTTGCTTCGCTAGAACCCTACTCTGTGCTGCGAGTGTCGCTGCTTCGTCTTCCTTC1800               CTCAAGTTCGATCTGATTGTGTGTGTGGGGGGGCGCAGGTAGGGCGAGGAGGGAGCCAAA1860               TCCAAATCAGCAGCCATGGCGCAGATGCTGCTCCATGGGACGCTGCACGCC1911                        MetAlaGlnMetLeuLeuHisGlyThrLeuHisAla                                           815820                                                                         ACCATCTTCGAGGCGGCGTCGCTCTCCAACCCGCACCGCGCCAGCGGA1959                           ThrIlePheGluAlaAlaSerLeuSerAsnProHisArgAlaSerGly                               825830835840                                                                   AGCGCCCCCAAGTTCATCCGCAAGGTTCGGACCCTTCTCCTTAATCTACTCGTC2013                     SerAlaProLysPheIleArgLys                                                       845                                                                            TTTGCTCTTGCTCTTTTTCTTTTGTGTCCCTTTCTTGTGTGTGCGTTTGCATGAGCCCGA2073               ATTTGATCTGCTAGTGCACAGTACAGTCAGATACACTGAAACGATCTGGAAATTCTGGAT2133               TATTAGGAAAAATAAAGAGGTAGTAGACAAGAATTGGAGATACTTTCTATCAAGATTGGT2193               CTATTATGCTTGGCCATTTCTTGTTTGACCCAAGTACTTCTTTGAATCTAGAGTTTGCTG2253               TGTGTGATGTGGTGTGTGTTTGTGTCACCAAAAATCTTCATTAGCTAAAACTGAAATTTT2313               ATTTATTAACTGACCTACTAAAAATGTAGAGTTCTCTGTGTGTGATGTGTGCTTGTGTCA2373               CCAAAAATCTTGATTTGATAGAGTTTTTATTTATTTATTAACTGACCTACTACAAATCTA2433               TTGCTGTATGCTATGTGTGTCTGTATCACCTGAAATGCAATGTCTTCTTCTTTGTTGTTC2493               TTGATCTAACACGTGAGCTCATGTCAACAGTTTGTGGAGGGGATTGAGGACACT2547                     PheValGluGlyIleGluAspThr                                                       15                                                                             GTGGGTGTCGGCAAAGGCGCCACCAAGGTGTATTCTACCATTGATCTG2595                           ValGlyValGlyLysGlyAlaThrLysValTyrSerThrIleAspLeu                               101520                                                                         GAGAAAGCTCGTGTAGGGCGAACTAGGATGATAACCAATGAGCCCATC2643                           GluLysAlaArgValGlyArgThrArgMetIleThrAsnGluProIle                               25303540                                                                       AACCCTCGCTGGTATGAGTCGTTCCACATCTATTGCGCTCATATGGCT2691                           AsnProArgTrpTyrGluSerPheHisIleTyrCysAlaHisMetAla                               455055                                                                         TCCAATGTGATCTTCACTGTCAAGATTGATAACCCTATTGGGGCAACG2739                           SerAsnValIlePheThrValLysIleAspAsnProIleGlyAlaThr                               606570                                                                         AATATTGGGAGGGCTTACCTGCCTGTCCAAGAGCTTCTCAATGGAGAG2787                           AsnIleGlyArgAlaTyrLeuProValGlnGluLeuLeuAsnGlyGlu                               758085                                                                         GAGATTGACAGA2799                                                               GluIleAspArg                                                                   90                                                                             (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetAlaGlnMetLeuLeuHisGlyThrLeuHisAlaThrIlePheGlu                               151015                                                                         AlaAlaSerLeuSerAsnProHisArgAlaSerGlySerAlaProLys                               202530                                                                         PheIleArgLys                                                                   35                                                                             (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 92 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        PheValGluGlyIleGluAspThrValGlyValGlyLysGlyAlaThr                               151015                                                                         LysValTyrSerThrIleAspLeuGluLysAlaArgValGlyArgThr                               202530                                                                         ArgMetIleThrAsnGluProIleAsnProArgTrpTyrGluSerPhe                               354045                                                                         HisIleTyrCysAlaHisMetAlaSerAsnValIlePheThrValLys                               505560                                                                         IleAspAsnProIleGlyAlaThrAsnIleGlyArgAlaTyrLeuPro                               65707580                                                                       ValGlnGluLeuLeuAsnGlyGluGluIleAspArg                                           8590                                                                           __________________________________________________________________________ 

We claim:
 1. A cloned DNA which encodes phospholipase D originated from a plant, wherein said DNA comprises a nucleotide sequence selected from the group consisting of nucleotides 182-2617 of SEQ ID NO:1 and nucleotides 107-2542 of SEQ ID NO:3 or a sequence complementary thereto or a sequence which specifically hybridizes to said cloned DNA or said complementary sequence in a hybridization solution containing 0.5M sodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA and 100 mg/ml of salmon sperm DNA at 65° C. for 16 hours and washing twice at 65° C. for twenty minutes in a washing solution containing 0.5×SSC and 0.1% SDS.
 2. The DNA according to claim 1, which encodes phospholipase D originated from a monocotyledonous plant.
 3. A cloned DNA which encodes an amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having the same sequence as shown in SEQ ID NO. 2 except that one or more amino acids are added, deleted or substituted, said amino acid sequence giving enzyme activity of phospholipase D.
 4. A cloned DNA which encodes the amino acid sequence shown in SEQ ID NO:
 2. 5. A cloned DNA which encodes an amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having the same sequence as shown in SEQ ID NO. 4 except that one or more amino acids are added, deleted or substituted, said amino acid sequence giving enzyme activity of phospholipase D.
 6. A cloned DNA which comprises a nucleotide sequence shown in SEQ ID NO:
 5. 7. The DNA according to claim 3, which has a nucleotide sequence shown in SEQ ID NO. 1 or has the same nucleotide sequence as shown in SEQ ID NO. 1 except that one or more nucleotides are added, deleted or substituted, said nucleotide sequence encodes an amino acid sequence giving enzyme activity of phospholipase D.
 8. The DNA according to claim 7, which has a nucleotide sequence shown in SEQ ID NO.
 1. 9. The DNA according to claim 5, which has a nucleotide sequence shown in SEQ ID NO. 3 or has the same nucleotide sequence as shown in SEQ ID NO. 3 except that one or more nucleotides are added, deleted or substituted, said nucleotide sequence encodes an amino acid sequence giving enzyme activity of phospholipase D.
 10. The DNA according to claim 9, which has a nucleotide sequence shown in SEQ ID NO.
 3. 11. The DNA according to claim 4, which has a nucleotide sequence from 182th to 2617th nucleotide in the nucleotide sequence shown in SEQ ID NO.
 1. 12. The DNA according to claim 6, which has a nucleotide sequence from 107th to 2542th nucleotide in the nucleotide sequence shown in SEQ ID NO.
 3. 13. A cloned DNA which regulates expression of phospholipase D gene originated from a plant.
 14. A cloned DNA which has a nucleotide sequence shown in SEQ ID NO. 5 or has the same nucleotide sequence as shown in SEQ ID NO. 5 except that one or more nucleotides are added, deleted or substituted, said nucleotide sequence regulates expression of the DNA encoding an amino acid sequence giving enzyme activity of phospholipase D.
 15. A cloned DNA which comprises a nucleotide sequence shown in SEQ ID NO:
 5. 16. A cloned DNA which comprises a nucleotide sequence from 1st to 1875th nucleotide in the nucleotide sequence shown in SEQ ID NO:
 5. 17. A cloned DNA which has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 or a sequence complementary thereto or a DNA which specifically hybridizes to said cloned DNA or said complementary sequence in a hybridization solution containing 0.5M sodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA and 100 μg/ml of salmon sperm DNA at 45° C. or 65° C. for 16 hours and washing twice at 45° C. or 650° C. for twenty minutes in a washing solution containing 0.3M NaCl and 30 mM sodium citrate or in a washing solution containing 0.5×SSC containing 0.1% SDS.
 18. An antisense polynucleotide which can suppress the expression of a DNA which encodes a phospholipase D originated from a plant, wherein said DNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 or wherein said DNA specifically hybridizes to said antisense sequence in a hybridization solution containing 0.5M sodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA and 100 μg/ml of salmon sperm DNA, at 45° C. or 65° C. for 16 hours and washing twice at 45° C. or 65° C. for twenty minutes in a washing solution containing 0.3M NaCl and 30 mM sodium citrate or in a washing solution containing 0.5×SSC containing 0.1% SDS.
 19. The cloned DNA of claim 17, wherein said plant is a rice plant.
 20. The cloned DNA of claim 17, wherein said plant is a corn plant.
 21. The cloned DNA of claim 17, which is a vector containing said DNA.
 22. The cloned DNA of claim 17, which is a cloning vector containing said DNA.
 23. A cell which contains the DNA of claim 17 or the vector of claim
 21. 